The long-term objective of this research project is to better understand the genetic basis for severe, potentially life-threatening toxicity secondary to treatment with 5-Fluorouracil (5-FU) and in particular further characterize the pharmacogenetic syndrome of dihydropyrimidine dehydrogenase (DPD) deficiency. During the next grant period we will further clarify the molecular mechanisms of regulation of the gene responsible for expression of DPD (DPYD), the role of the ubiquitin-proteasome system in DPD protein turnover, develop phenotypic and genotypic diagnostic tests for DPD deficiency, and lastly examine the role of other enzymatic steps in 5-FU metabolism in the cause of severe 5-FU toxicity. In approaching each of the specific aims listed below, we will continue to obtain and utilize biochemical and molecular data (e.g., DPD enzyme activity, DPD mRNA level, and analysis for mutations in DPYD gene) from patients presenting with grade IV toxicity after 5-FU therapy. The specific aims will include: Spec. Aim 1) - Identify and clarify the role of additional transcriptional regulatory elements affecting DPYD gene expression including identification of a.) transcription factor(s) that bind to regulatory elements I and II in the previously identified promoter, and b) additional potential regulatory regions in intron 1 and the 3'-untranslated region; Spec. Aim 2) - Determine the role of the ubiquitin (Ub) proteasome system in the regulation of DPD protein - a) determine DPD protein half-life for wild type and mutant DPD protein, and b) identify putative destabilizing element(s) of DPD protein; Spec. Aim 3) - Develop user-friendly diagnostic tests for DPD deficiency including a) phenotypic tests of DPD deficiency suitable for routine screening, and b) genotypic tests for specific causes of DPD deficiency; and Spec. Aim 4) - Determine the role of other factors that may contribute to severe 5-FU toxicity including a) altered gene expression of the 5-FU site of action - thymidylate synthase, b) altered gene expression of anabolic enzymes, e.g. uridine and thymidine phosphorylases and kinases and orotate phosphoribosyltransferase, and c) altered gene expression of other catabolic enzymes, e.g. dihydropyrimidinase. Successful progress on this research project should translate into improved care for patients receiving fluoropyrimidine drugs in the future.